The present invention relates generally to novel formulations comprising factor IX.
A variety of factors involved in the blood clotting process have been identified, including factor IX, a plasma glycoprotein. A deficiency of factor IX characterizes a type of hemophilia (type B). Treatment of this disease has traditionally involved intra venous infusion of human plasma-derived protein concentrates of factor IX. Infusion of blood concentrates involves the risk of transmission of various infectious agents, such as viral hepatitis and HIV, or thromboembolic factors. An alternative method of producing factor IX, by recombinant DNA techniques, has been described in U.S. Pat. No. 4,770,999, Kaufman et al., Sep. 13, 1988. The cDNA coding for human factor IX has been isolated, characterized, and cloned into expression vectors. See, for example, Choo et al., Nature 299:178-180 (1982); Fair et al., Blood 64:194-204 (1984); and Kurachi et al., Proc. Natl. Acad. Sci., U.S.A. 79:6461-6464 (1982). Thus, through advances in recombinant DNA technology, it has been possible to produce factor IX protein.
It is desirable to have concentrated forms of bulk protein, e.g., factor IX, which, in turn, may be stored and which are suitable for further manufacture of finished dosage forms of protein. Typically, a purification process for a protein results in concentrating the protein. This concentrated protein, also known as bulk protein, may be in a formulation buffer. Bulk protein, typically at a concentration of about 2 to at least 20 mg/ml, can then be shipped frozen to a fill/finish facility where it is diluted to an appropriate dosage concentration and placed into dosage vials. These diluted samples can be lyophilized, i.e., freeze-dried. The lyophilized samples may be kept in long-term storage and reconstituted at a later time by adding a suitable administration diluent just prior to patient use.
Protein stability can be affected inter alia by such factors as ionic strength, pH, temperature, repeated cycles of freeze/thaw and exposures to shear forces. Active protein may be lost as a result of physical instabilities, including denaturation and aggregation (both soluble and insoluble aggregate formation), as well as chemical instabilities, including, for example, hydrolysis, deamidation and oxidation, to name just a few. For a general review of stability of protein pharmaceuticals, see, for example, Manning, et al., Pharmaceutical Research 6:903-918 (1989).
While the possible occurrence of protein instabilities is widely appreciated, it is impossible to predict particular instability problems of a particular protein. Any of these instabilities can result in the formation of a protein, protein by-product, or derivative having lowered activity, increased toxicity, and/or increased immunogenicity. Indeed, protein precipitation may lead to thrombosis, non-homogeneity of dosage form and amount, as well as clogged syringes. Also, specific to factor IX, there are several post-translational modifications (for example, the gamma carboxylation of certain glutamic acid residues in the N-terminus and the addition of carbohydrate) which may be important in maintaining biological activity and which may be susceptible to modification upon storage. Thus, the safety and efficacy of any pharmaceutical formulation of a protein is directly related to its stability.
In addition to stability considerations, one generally selects excipients which are or will meet with the approval of various world-wide medical regulatory agencies. The solution should be isotonic and the pH in a physiologically suitable range. The choice and amount of buffer used is important to achieve the desired pH range. Moreover, in the case of factor IX, agents such as xe2x80x9cheparinxe2x80x9d are to be avoided because of potential interference with clotting time assay analysis and with accurate assessment of thrombogenic potential.
Currently, there are only two commercially available, carrier-protein-free, plasma-derived factor IX formulations. Alpha Therapeutic Corporation provides lyophilized AlphaNine(copyright) SD: comprising heparin, dextrose, polysorbate 80, and tri(n-butyl) phosphate. This preparation is meant to be stored at temperatures between 2xc2x0 and 8xc2x0 C. As noted supra, heparin is to be avoided as it is an anti-coagulant and tri(n-butyl) phosphate is irritating to mucous membranes; thus, this formulation is less than ideal. Armour Pharmaceutical Company""s lyophilized Mononine(copyright): comprising histidine, sodium chloride and mannitol is similarly meant to be stored at 2xc2x0 to 8xc2x0 C. The package insert recommends not storing this formulation for greater than one month at room temperature.
Ideally, formulations developed should also be stable for factor IX bulk storage in high concentration (xe2x89xa620 mg/ml, for example) which allows for relatively small volumes for fill/finish at the appropriate dose and also allows for alternate methods of administration which may require high protein concentration, e.g., sub cutaneous administration. Accordingly, there continues to exist a need in the art for methods for improving factor IX protein stability (and maintaining activity levels) during the concentration process, and the lyophilization process, as well as providing stable formulations during prolonged storage.
One aspect of the present invention provides novel compositions and methods for providing concentrated preparations of factor IX, useful as bulk drug product. These compositions, either frozen, liquid, or lyophilized, comprise factor IX, a bulking agent, such as glycine, and a cryoprotectant. A preferred factor IX concentration ranges from about 0.1 to at least 20 mg/ml (equivalent to about 20 to at least 4000 U/ml). Preferred bulking agents include glycine, and/or a magnesium, calcium, or chloride salt, preferably ranging in concentration from about 0.5 to 300 mM. Suitable cryoprotectants include polyols, such as mannitol and sucrose, and preferably range in concentration from about 0.5 to 2%. Optionally, these bulk drug product compositions may also contain a surfactant or detergent, such as polysorbate (e.g., Tween-80) or polyethyleneglycol (PEG), which may also serve as a cryoprotectant during the freezing step. The surfactant preferably ranges from about 0.005 to 0.05%. Preferably, the concentrations of the excipients provide a combined osmolality of about 250 to 350 milliosmolal (mOsM), preferably about 300 mOsMxc2x150 mOsM, and further, may contain an appropriate buffering agent to maintain a physiologically suitable pH e.g., in the range preferably of about 6.0 to 8.0. Buffering agents preferably include histidine, and sodium or potassium phosphate, with a target pH of about 6.5 to 7.5, all at about 5-50 mM.
Another aspect of the present invention provides formulations of factor IX suitable for administration in a final dosage form, for example, via intra venous or sub cutaneous injection. Preferred formulations include factor IX concentrations ranging from about 0.1 to at least 20 mg/ml, about 0.5 to 2% sucrose, about 0.1 to 0.3 M glycine, and about 0.005% to 0.02% polysorbate, with histidine as a buffering agent, ranging from about 5 to 50 mM. A preferred lyophilized formulation comprises about 0.1 to at least 10 mg/ml factor IX, about 260 mM glycine, about 1% sucrose, about 0.005% polysorbate, and about 10 mM histidine, at pH 7.0.
As used herein, the terms lyophilization, lyophilized, and freeze-dried include but are not limited to processes including xe2x80x9cfreezingxe2x80x9d a solution followed by xe2x80x9cdryingxe2x80x9d, optionally in vacuo. As used herein, the term xe2x80x9cbulking agentxe2x80x9d comprises agents which provide good lyophilized cake properties, which help the protein overcome various stresses (shear/freezing for example) associated with the lyophilization process, and which help to maintain protein activity levels. Exemplary bulking agents include, but are not limited to, glycine, MgCl2, CaCl2, NaCl, and the like. These agents contribute to the tonicity of the formulations. Cryoprotectants also contribute to the tonicity. The term xe2x80x9ccryoprotectantsxe2x80x9d generally includes agents which provide stability to the protein from freezing-induced stresses; however, the term also includes agents that provide stability, e.g., to bulk drug formulations during storage from non-freezing-induced stresses. Exemplary cryoprotectants include polyols, and include saccharides such as sucrose and mannitol, as well as including surfactants such as polysorbate, or polyethyleneglycol, and the like. The term xe2x80x9clyoprotectantxe2x80x9d includes agents that provide stability to the protein during water removal from the system during the drying process, presumably by maintaining the proper conformation of the protein through hydrogen bonding. Cryoprotectants can also have lyoprotectant effects. While preferred concentrations of cryoprotectant range from about 0.5 to 2%, relatively high concentrations, for example 5%, are suitable with the levels used limited only by those customarily used in clinical practice.
xe2x80x9cSurfactantsxe2x80x9d generally include those agents which protect the protein from air/solution interface induced stresses and solution/surface induced stresses (e.g., resulting in protein aggregation), and may include detergents such as polysorbate-80 (Tween), for example, 0.005-0.05% (weight/volume), or polyethyleneglycol (PEG), such as PEG8000, for example. Optionally, relatively high concentrations, e.g., up to 0.5%, are suitable for maintaining protein stability; however, the levels used in actual practice are customarily limited by clinical practice.
The term xe2x80x9cbuffering agentxe2x80x9d encompasses those agents which maintain the solution pH in an acceptable range prior to lyophilization and may include histidine, phosphate (sodium or potassium), tris(tris(hydroxymethyl)aminomethane), diethanolamine, and the like. The upper concentration limits are generally higher for xe2x80x9cbulkxe2x80x9d protein than for xe2x80x9cdosagexe2x80x9d protein forms as is readily appreciated by one skilled in the art. For example, while buffer concentrations can range from several millimolar up to the upper limit of their solubility, e.g., histidine could be as high as 200 mM, one skilled in the art would also take into consideration achieving/maintaining an appropriate physiologically suitable concentration. Percentages are weight/weight when referring to solids and weight/volume when referring to liquids. The term xe2x80x9cisotonic,xe2x80x9d 300xc2x150 mOsM, is meant to be a measure of osmolality of the protein solution prior to lyophilization; reconstitution is typically with water for injection (WFI). Maintaining physiological osmolality is important for the dosage formulations. However, for bulk formulations, much higher concentrations can be effectively utilized as long as the solution is made isotonic prior to use. The term xe2x80x9cexcipientsxe2x80x9d includes pharmaceutically acceptable reagents to provide good lyophilized cake properties (bulking agents) as well as provide lyoprotection and cryoprotection of the protein, maintenance of pH, and proper conformation of the protein during storage so that substantial retention of biological activity (protein stability) is maintained.
As used herein, factor IX concentration is conveniently expressed as mg/ml or as U/ml, with 1 mg approximately equal to 200 U/mlxc2x1100 U/ml.